Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 3, 2011, pp. 269-274

Indian Journal of Medical Microbiology, Vol. 29, No. 3, July-September, 2011, pp. 269-274

Original Article

OXA beta-lactamase-mediated carbapenem resistance in Acinetobacter baumannii

SM Amudhan¹, U Sekar¹, K Arunagiri², B Sekar³

¹ Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Porur, Chennai, India
² Department of Molecular Biology, Department of Microbiology, Central Leprosy Teaching and Research Institute, Chengalpattu, India
³ Pasteur Institute of India, Coonoor, Nilgiris, Tamil Nadu, India
Correspondence Address: U Sekar, Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Porur, Chennai, India, umasekar02@yahoo.co.in

Date of Submission: 21-Apr-2011 
Date of Acceptance: 01-Jul-2011

Code Number: mb11065

PMID: 21860108

DOI: 10.4103/0255-0857.83911

Abstract

Keywords: Acinetobacter baumannii, bla_OXA , carbapenamases, modified Hodge test, minimum inhibitory concentration

Introduction

Multidrug resistant Acinetobacter baumannii is a significant pathogen in health care settings, where it causes a multitude of infections that include bacteremia, pneumonia, meningitis and urinary tract and wound infections. Its ability to survive under a wide range of environmental conditions makes it a frequent cause of outbreaks of infection and an endemic health care-associated pathogen. ^[1] A. baumannii is often resistant to a wide variety of antimicrobial agents, including carbapenems. Carbapenem resistance in A. baumannii is due to a variety of combined mechanisms such as hydrolysis by beta-lactamases, alterations in the outer membrane protein and penicillin-binding proteins and increased activity of efflux pumps. Acquired resistance to carbapenems, mediated by the Ambler class D beta-lactamases or OXA-type carbapenamases and Ambler class B metallo-beta-lactamases are of greatest concern as they are encoded by genes which are transmissible and account for most of the resistance to carbapenems. ^[2],[3] Carbapenem-hydrolyzing bla_OXA-23 was first reported in A. baumannii in 1985. Since then several of them have been reported worldwide. Four families of OXA genes have been identified in A. baumannii: bla_OXA-23 like (bla_OXA-23 , bla_OXA-27 and bla _OXA-49 ); bla_OXA-24 like (bla_OXA-24 , bla_OXA-25 , bla_OXA-26 and bla_OXA-40 ); bla_OXA-58 and bla_OXA-51 like. The last group constitutes a family of chromosomal enzymes typically present in A. baumannii.^[2],[4],[5] Though the presence of bla_OXA genes in A. baumannii is widely known, there is a paucity of information on the distribution of different types of OXA carbapenamases in isolates from the Indian subcontinent. ^[6],[7],[8] This study was undertaken to detect the prevalence of different bla_OXA -type carbapenamases among nosocomial isolates of A. baumannii. The simultaneous presence of metallo-beta-lactamases (bla_VIM and bla_IMP ) type of carbapenamases was also determined in the study isolates.

Materials and Methods

Bacterial strains

The study was conducted in a 1600-bedded university teaching hospital from April 2010 to October 2010. It included 116 clinically significant, non-duplicate, carbapenem resistant Acinetobacter baumannii recovered from clinical specimens of patients hospitalized for >48 h. The isolates were obtained from clinical specimens such as blood, cerebrospinal fluid, pus, wound swabs, and urine and lower respiratory secretions (bronchoalveolar lavage, bronchial wash and endotracheal secretions). The organisms were identified up to the species level using Microscan Walk Away 96, Gram-negative panels. Care was taken to differentiate commensals from pathogens for isolates obtained from nonsterile sites (respiratory tract, urinary tract, and wound swabs). The significance of the isolates was based on clinical history, presence of the organism in the Gram stain, presence of intracellular forms of the organism, and pure growth in culture with a significant colony count.

Antimicrobial susceptibility testing

Susceptibility to various classes of antibiotics was determined by the disc diffusion method in accordance with Clinical Laboratory Standard Institute (CLSI) guidelines. ^[6] The antibiotics tested were amikacin (30 μg), ciprofloxacin (5 μg), ceftazidime (30 μg), piperacillin-tazobactam (100/10 μg), imipenem (10 μg), meropenem (10 μg) and polymyxin B (300 units), ^[9] from Himedia Laboratories (Mumbai, Maharashtra, India). Disc diffusion susceptibility testing was also performed for all the isolates using tigecycline disks (15 μg; BBL ^TM BD, USA). The interpretation of zone diameters was done using the United States Food and Administration′s tigecycline susceptibility breakpoint criteria listed for Enterobacteriaceae (susceptible ΃19 mm, intermediate 15-18 mm, resistance ≤14 mm). ^[10] MIC to imipenem and meropenem was done by the agar dilution method (range: 0.008-256 μg/ml) in accordance with CLSI guidelines. ^[9]

Detection of carbapenamases

The detection of carbapenamases was done by the modified Hodge Test. Escherichia coli ATCC 25922 was cultured overnight and suspended to achieve a 0.5 McFarland standard turbidity and was lawn cultured onto a Mueller-Hinton agar plate using a sterile cotton swab. After drying, a disk containing imipenem (10 μg) was placed at the center of the plate, and an overnight cultured test strain was heavily streaked from the center to the periphery of the plate. The presence of a distorted zone after overnight incubation was interpreted as a positive result. ^[11] Though CLSI ^[9] does not advocate the use of the modified Hodge test for the detection of carbapenamase production in nonfermenting Gram-negative bacilli, several authors have found the modified Hodge test using imipenem as a useful screening test for carbapenamase production. ^{[12],[13],[14]}

Detection of metallo-beta-lactamases

Zone enhancement with ethylenediaminetetraacetic acid (EDTA)-impregnated imipenem and ceftazidime discs

Test organisms were inoculated on the plates with Mueller-Hinton agar. A 0.5 M EDTA solution was prepared by dissolving 186.1 g of disodium EDTA in 1000 ml of distilled water and adjusting its pH to 8.0. The EDTA solution was sterilized by autoclaving. Two 10-μg imipenem discs and two 30-μg ceftazidime discs were placed on the surface of the agar plate and EDTA solution (5 μl) was added to one of them to obtain a desired concentration of 750 μg. The inhibition zones of the imipenem and ceftazidime and their EDTA-impregnated discs were compared after incubation in air at 37°C. A zone size difference of ΃7 mm was taken as indicative of metallo-beta-lactamase production. ^[15]

Detection of carbapenem resistance genes

DNA template

A 1:10 dilution of an overnight culture was boiled for 10 mins. Amplification was then performed with 10 μl of this dilution as the DNA template.

Multiplex PCR for the detection of OXA genes ^[5]

Multiplex PCR was done for the detection of the four families of OXA-type carbapenamases found in A baumannii. Sequences of primers used for multiplex PCR for the detection of genes encoding bla_OXA-23 like, bla_OXA-24 like, bla_OXA-51 like and bla_OXA-58 like genes are given in [Table - 1].

The PCR conditions were as follows: Initial denaturation at 94°C for 5 min, 33 cycles of 94°C for 25 s, 53°C for 40 s and 72°C for 50 s, followed by an elongation step at 72°C for 6 min.

The PCR products of 501 bp (bla_OXA-23 like), 353 bp (bla_OXA-51 like), 246 bp (bla_OXA-24 like) and 599 bp (bla_OXA-58 like) were visualized by agarose gel electrophoresis.

PCR for metallo-beta-lactamase genes: bla_VIM and bla_IMP ^[16] Primers used are given in [Table - 1].

PCR conditions included 30 cycles of amplification under the following conditions: Denaturation at 95°C for 30 s, annealing for 1 min at specific temperatures (blaVIM , 66°C, and bla_IMP , 45°C), and extension at 72°C for 1 min/kb product. Cycling was followed by a final extension at 72°C for 10 min. The PCR product of 500 bp (bla_VIM ) and 432 bp (bla_IMP ) was visualized by agarose gel electrophoresis.

DNA sequencing

The PCR products of representative isolates were then purified by using the PCR DNA purification kit (QIA Quick Gel Extraction Kit; Qiagen, Valencia, CA, USA) and subjected to automated DNA sequencing (ABI 3100, Genetic Analyser; Applied Biosystems, Foster City, CA, USA). The aligned sequences were then analysed with the Bioedit sequence program and similarity searches for the nucleotide sequences were performed with the BLAST program (http://www.ncbi.nlm.nih.gov).

Results

A. baumannii were isolated from clinical specimens such as blood (n = 25), respiratory secretions (n = 62), pus and wound swab (n = 18), cerebrospinal fluid (n = 4), body fluids (n = 3) and urine (n = 4). Most isolates were from patients in the intensive care units (ICU) of the health care facility (n = 111) and only 5 were from patients in the wards.

All of them were resistant to amikacin, ciprofloxacin, ceftazidime, piperacillin-tazobactam, imipenem and meropenem. The MIC to imipenem and meropenem ranged from 8 to 128 μg/ml. The MIC₅₀ and MIC₉₀ values for imipenem were 16 and 32 μg/ml, respectively. For meropenem, MIC₅₀ and MIC₉₀ values were 32 and 64 μg/ml, respectively.

Among the 116 isolates, 97.4% (n = 113) were susceptible to polymyxin and 93.1% (n = 108) to tigecycline.

Phenotypic tests

Of 116 A. baumannii, the modified Hodge test was positive for 113 (97.4%) of isolates [Figure - 1] while in 3, the test was negative. The metallo-beta-lactamase screening test with EDTA was positive in 92 (79.3%) isolates.

PCR

bla_OXA-23 like, bla_OXA-24 like, bla_OXA-51 like and bla_OXA-58 like were detected in the majority (n = 106) of isolates [Figure - 2]. Most of them carried the bla_OXA-51 like (n = 99) and bla_OXA-23 like (n = 95) genes. Both coexisted in 89 isolates. Of the other OXA types, bla_OXA-24 like was found only in two isolates, of which one also harboured bla_OXA-51 like and bla _OXA-58 like genes. The distribution of different types of OXA genes in the test organisms is given in [Table - 2].

Among the modified Hodge test-positive A. baumannii (n = 113), OXA genes were not detected in seven isolates. Of them (n = 7), bla_VIM was detected in four and the remaining three did not have bla_OXA /bla_VIM or bla_IMP.

Regarding metallo-beta-lactamase, PCR detected bla_VIM in 54 isolates, of which 1 carried both bla_VIM and bla_IMP [Figure - 3] and [Figure - 4]. Of the above, 50 also harboured the OXA gene, particularly the bla_OXA-23 like and bla_OXA-51 like.

On review of the patient care areas from where the bla_OXA -positive (n = 106) isolates were obtained, it was found that the multidisciplinary open ICU had the highest number of isolates carrying the bla_OXA gene (69/74), followed by neurosurgery ICU (25/28), cardiothoracic ICU (6/7) and general wards (4/5). From the paediatric ICU, though the isolates obtained were only two, both carried the bla_OXAgene.

Discussion

A. baumannii accounts for a substantial proportion of endemic nosocomial infections. Multidrug resistance increasingly reported in these pathogens is posing a threat to hospitalized patients due to the limitation of therapeutic options. The acquisition of multidrug resistance is related to environmental contamination and contact with transiently colonized health care providers. Carbapenems have been the drug of choice for treatment of infections caused by A. baumannii. However, in recent years, the number of isolates showing resistance to carbapenems has increased worldwide. ^{[2],[12],[17]} This is mediated by the lack of drug penetration (i.e. porin mutations and efflux pumps) and/or carbapenem-hydrolyzing beta-lactamase enzymes such as OXA carbapenamases and metallo-beta-lactamases. ^[2]

The first identified OXA-type enzyme with carbapenem-hydrolyzing activity was from an A. baumannii strain isolated in 1985 from Scotland and was originally named ARI-1, but was renamed as bla_OXA-23 . The bla_OXA-23 cluster (bla_OXA-23,27,49 ) now contributes to carbapenem resistance in A. baumannii globally. Two other plasmid-encoded acquired OXA-type clusters with a carbapenamase activity have been described, which are bla_OXA-24 -like (bla_{OXA-24,25,26,40} ) and bla_OXA-58 -like genes. The bla_OXA-23 , bla_OXA-24 and bla_OXA-58 like enzymes are plasmid/chromosomally encoded which explains their widespread distribution. The bla_OXA-51 like gene cluster is unique in that it naturally occurs in A.baumannii. Therefore it is chromosomally located and is widely prevalent. Similar to other class D enzymes, they have a greater affinity for imipenem than meropenem. Their role in carbapenem resistance is related to the presence of an insertion sequence ISAba1, situated upstream, possibly providing a promoter for the hyperproduction of beta-lactamase genes. ^{[2],[18],[19]} Strains that harbour multiple OXA encoding genes have been reported from several geographical areas. ^{[12],[17],[20]}

In this study, overall OXA carbapenamases were detected in 91.3% (n = 106) of carbapenem resistant A. baumannii. bla_OXA-51 (n = 99) and bla_OXA-23 (n = 95) were the most common OXA carbapenamases and they coexisted in 89 isolates. bla_OXA-51 was found alone in nine isolates and bla_OXA-23 alone in six isolates. bla_OXA-24 was detected only in two isolates, of which one also carried bla_OXA-51 and bla_OXA-58 .

Numerous studies have reported that bla_OXA-23 is the most frequent type of carbapenamases identified among the carbapenem resistant A. baumannii.^{[5],[13],[19]} The coexistence of bla_OXA-23 like, bla_OXA-24 like, bla_OXA-51 like and bla_OXA-58 like genes has been reported, especially that of bla_OXA-23 and bla_OXA-51. ^[14],[20] Bla_OXA-24 has been reported from Spain, Belgium, France and United States while bla_OXA-58 has been identified in many countries. ^[2] There is a paucity of information on the frequency of occurrence, prevalence and distribution of OXA carbapenamases from India. ^[6],[7],[8] In one study on 62 isolates of the Acinetobacter species, bla_OXA-23 was the most common. ^[6] In another study on four carbapenem resistant A. baumannii blood stream isolates, bla_OXA-23 was found to be the most prevalent. ^[7]

Among the 54 bla_VIM -positive isolates, 1 also carried bla_IMP . The metallo-beta-lactamase screen test was positive in 92 isolates, thereby suggesting the presence of other metallo-beta-lactamases such as GIM, SPM and NDM-1. ^[1],[8] Despite metallo-beta-lactamases being less commonly identified in A. baumannii compared to the OXA carbapenamases, their hydrolytic activity towards carbapenems are significantly more potent. These enzymes hydrolyze all beta-lactams except the monobactams (aztreonam). ^[1],[4] In this study, bla_VIM coexisted with bla_OXA-23 and bla_OXA-51 in 49 isolates and in 1 isolate bla_VIM , bla_IMP , bla_OXA-23 and bla_OXA-51 were found. The simultaneous existence of bla_OXA and metallo-beta-lactamases has been reported from several geographic regions. ^{[2],[8],[14],[19]}

In four carbapenem resistant A. baumannii, bla_VIM was detected without the OXA genes. Among the modified Hodge test-positive isolates (n = 113), PCR for OXA, bla_VIM and bla_IMP was negative in three isolates suggesting the presence of other carbapenamases.

For the carbapenem resistant isolates which were modified Hodge test-negative (n = 3), it may be assumed to be due to the presence of porin mutations and upregulation of efflux pumps.

Among the 116 study isolates, 97.4% (n = 113) were susceptible to polymyxin and 93.1% (n = 108) to tigecycline. The lack of universal susceptibility to these two drugs is a cause for concern. All the study isolates were uniformly resistant to aminoglycosides and fluoroquinolones.

Though carbapenems are the drug of choice to treat A. baumannii infections, such resistance profiles limit therapeutic options to polymyxins and tigecycline. These drugs also have their own limitations and are not indicated as the drug of choice under a variety of clinical conditions. Easy and simple phenotypic tests are required for the early identification of carbapenamases in the clinical laboratories to notify the treating physicians and also devise methods to contain their spread.

Since the acquisition of multidrug resistant A. baumannii is related to environmental contamination and colonization in health care providers, control measures should address the source of infection. Continued careful attention to hand hygiene, contact isolation, barrier precautions, adequate environmental cleaning, and careful disinfection of patient care equipments along with surveillance are essential to prevent the outbreak of infections caused by these multidrug resistant strains. ^[21]

To conclude, bla_OXA-23 and bla_OXA-51 are the most common types of OXA carbapenamases in A. baumannii. This type of resistance is a factor with a significant threat in hospitals. It should be addressed with alternative and newer therapeutic strategies, strict infection control measures and continuous surveillance. A simultaneous existence of different classes of carbapenamases is a problem to reckon with and hence detection methods are required for each of these. In outbreak settings, an initial screening of the putative carbapenamase producers will help to organize intervention and early directed therapy.

References

Copyright 2011 - Indian Journal of Medical Microbiology

The following images related to this document are available:

Photo images